Fig. 3

ORF3a interacts with both N- and C-terminal fragments of human STING, and has no effect on cGAS-STING triggered IRF3 activation. a Schematic of the STING functional domains and various STING truncation constructs. b Identification of the regions in STING that are important for the ORF3a interaction. HEK293T cells were co-transfected with the ORF3a expression vector and various STING mutant constructs or control vector. Cell lysates were prepared and immunoprecipitated using anti-Flag beads 24 h after transfection. Precipitated samples were separated by SDS-PAGE, transferred to nitrocellulose membranes, and reacted with anti-HA antibody to detect ORF3a-HA and anti-Flag antibody to detect STING truncation constructs. Tubulin was used as the loading control. c The N- and C-terminal of STING are required for the interaction with ORF3a. HEK293T cells were co-transfected with ORF3a expression vector and the wild-type or truncated form of STING. Cell lysates were prepared and immunoprecipitated using anti-HA beads 24 h after transfection. Precipitated samples were separated by SDS-PAGE, transferred to nitrocellulose membranes, and reacted with anti-GFP antibody to detect full-length STING or its truncation constructs and anti-HA antibody to detect ORF3a-HA. GAPDH was used as the loading control. The bands of STING and its truncation were labeled with an asterisk. d ORF3a impairs IκBα degradation induced by cGAS-STING, but has no effect on cGAS-STING-triggered IRF3 and TBK1 phosphorylation. HEK293T cells were co-transfected with STING-Flag, Myc-cGAS, and ORF3a-HA or control vector. After 24 h, total cells were harvested, and the protein expression levels were analyzed by immunoblotting with anti-TBK1-p, anti-TBK1, anti-IRF3-p, anti-IRF3, anti-IκBα, anti-Flag, anti-Myc, anti-HA, or anti-GAPDH antibodies. e E1A and vIRF1 inhibit the expression of cGAS-STING-induced IRF3 phosphorylation. HEK293T cells were co-transfected with STING-Flag, Myc-cGAS, E1A-HA, and vIRF1-HA or control vector. After 24 h, total cells were harvested, and the protein expression levels were analyzed by immunoblotting with anti-IRF3-p, anti-Flag, anti-HA, or anti-GAPDH antibodies. f E1A and vIRF1, but not ORF3a, inhibit IRF3 response element activity induced by cGAS-STING. HEK293T cells were co-transfected with IRF3-Luc, cGAS, and STING expression vectors, and increasing amounts of ORF3a, E1A, or vIRF1 expression vectors. Transactivation of the luciferase reporter was determined as described before (n = 3 independent biological experiments). Luciferase activity stimulated by cGAS-STING alone was used as a positive control and set to 100%. Means and standard deviations are presented. Statistical significance was determined by two-sided unpaired t test, ***p < 0.001; ****p < 0.0001; NS, no significance